Fibrin is a natural gel with several biomedical applications. Fibrin gel has been used as a sealant because of its ability to bind to many tissues and its natural role in wound healing. Some specific applications include use as a sealant for vascular graft attachment, heart valve attachment, bone positioning in fractures and tendon repair (Sierra, D. H., Journal of Biomaterials Applications, 7:309-352, 1993). Additionally, these gels have been used as drug delivery devices, and for neuronal regeneration (Williams, et al., Journal of Comparative Neurobiology, 264:284-290, 1987).
The process by which fibrinogen is polymerized into fibrin has also been characterized. Initially, a protease cleaves the dimeric fibrinogen molecule at the two symmetric sites. There are several possible proteases that can cleave fibrinogen, including thrombin, reptilase, and protease III, and each one severs the protein at a different site (Francis, et al., Blood Cells, 19:291-307, 1993). Each of these cleavage sites have been located (FIG. 1). Once the fibrinogen is cleaved, a self-polymerization step occurs in which the fibrinogen monomers come together and form a non-covalently crosslinked polymer gel (Sierra, 1993). A schematic representation of the fibrin polymer is shown in FIG. 2. This self-assembly happens because binding sites become exposed after protease cleavage occurs. Once they are exposed, these binding sites in the center of the molecule can bind to other sites on the fibrinogen chains, these sites being present at the ends of the peptide chains (Stryer, L. In Biochemistry, W. H. Freeman and Company, N.Y., 1975). In this manner, a polymer network is formed. Factor XIIIa, a transglutaminase activated from factor XIII by thrombin proteolysis, may then covalently cross-link the polymer network. Other transglutaminases exist and may also be involved in covalent crosslinking and grafting to the fibrin network.
Once a crosslinked fibrin gel is formed, the subsequent degradation is tightly controlled. One of the key molecules in controlling the degradation of fibrin is xcex12-plasmin inhibitor (Aoki, N., Progress in Cardiovascular Disease, 21:267-286, 1979). This molecule acts by crosslinking to the xcex1 chain of fibrin through the action of factor XIIIa (Sakata, et al., Journal of Clinical Investigation, 65:290-297, 1980). By attaching itself to the gel, a high concentration of inhibitor can be localized to the gel. The inhibitor then acts by preventing the binding of plasminogen to fibrin (Aoki, et al., Thrombosis and Haemostasis, 39:22-31, 1978) and inactivating plasmin (Aoki, 1979). The xcex1-2 plasmin inhibitor contains a glutamine substrate. The exact sequence has been identified as NQEQVSPL (SEQ ID NO: 15), with the first glutamine being the active amino acid for crosslinking.
The components required for making fibrin gels can be obtained in two ways. One method is to cryoprecipitate the fibrinogen from plasma. In this process, factor XIII precipitates with the fibrinogen, so it is already present. The proteases are purified from plasma using similar methods. Another technique is to make recombinant forms of these proteins either in culture or with transgene animals. The advantage of this is that the purity is much higher, and the concentrations of each of these components can be controlled.
Cells interact with their environment through protein-protein, protein-oligosaccharide and protein-polysaccharide interactions at the cell surface. Extracellular matrix proteins provide a host of bioactive signals to the cell. This dense network is required to support the cells, and many proteins in the matrix have been shown to control cell adhesion, spreading, migration and differentiation (Carey, Annual Review of Physiology, 53:161-177, 1991). Some of the specific proteins that have shown to be particularly active include laminin, vitronectin, fibronectin, fibrin, fibrinogen and collagen (Lander, Journal of Trends in Neurological Science, 12:189-195, 1989). Many studies of laminin have been conducted, and it has been shown that laminin plays a vital role in the development and regeneration of nerves in vivo and nerve cells in vitro (Williams, Neurochemical Research, 12:851-869, 1987; Williams, et al., 1993), as well as in angiogenesis.
Some of the specific sequences that directly interact with cellular receptors and cause either adhesion, spreading or signal transduction have been identified. This means that the short active peptide sequences can be used instead of the entire protein for both in vivo and in vitro experiments. Laminin, a large multidomain protein (Martin, Annual Review of Cellular Biology, 3:57-85, 1987), has been shown to consist of three chains with several receptor-binding domains. These receptor-binding domains include the YIGSR (SEQ ID NO: 1) sequence of the laminin B1 chain ( Graf, et al., Cell, 48:989-996, 1987; Kleinman, et al., Archives of Biochemistry and Biophysics, 272:39-45, 1989; and Massia, et al., J. of Biol. Chem., 268:8053-8059, 1993), LRGDN (SEQ ID NO: 2) of the laminin A chain (Ignatius, et al., J. of Cell Biology, 111:709-720, 1990) and PDGSR (SEQ ID NO: 3) of the laminin B1 chain (Kleinman, et al., 1989). Several other recognition sequences for neuronal cells have also been identified. These include IKVAV (SEQ ID NO: 4) of the laminin A chain (Tashiro, et al., J. of Biol. Chem., 264:16174-16182, 1989) and the sequence RNIAEIIKDI (SEQ ID NO: 5) of the laminin B2 chain (Liesi, et al., FEBS Letters, 244:141-148, 1989). The receptors that bind to these specific sequences have also often been identified. A subset of cellular receptors that has shown to be responsible for much of the binding is the integrin superfamily (Rouslahti, E., J. of Clin. Investigation, 87:1-5, 1991). Integrins are protein heterodimers that consist of xcex1 and xcex2 subunits. Previous work has shown that the tripeptide RGD binds to several xcex21 and xcex23 integrins (Hynes, R. O., Cell, 69:1-25, 1992; Yamada, K. M., J. of Biol. Chem., 266:12809-12812, 1991), IKVAV(SEQ ID NO: 4) binds to a 110 kDa receptor (Tashiro, et al., J. of Biol. Chem., 264:16174-16182, 1989; Luckenbill-Edds, et al., Cell Tissue Research, 279:371-377, 1995), YIGSR (SEQ ID NO: 1) binds to a 67 kDa receptor (Graf, et al., 1987) and DGEA (SEQ ID NO: 6), a collagen sequence, binds to the xcex12,xcex21 integrin (Zutter and Santaro, Amer. J. of Pathology, 137:113-120, 1990). The receptor for the RNIAEIIKDI (SEQ ID NO: 5) sequence has not been reported.
Work has been done in crosslinking bioactive peptides to large carrier molecules and incorporating them within fibrin gels. By attaching the peptides to the large carrier polymers, the rate of diffusion out of the fibrin gel will be slowed down. In one series of experiments, polyacrylic acid was used as the carrier polymer and various sequences from laminin were covalently bound to them to confer neuroactivity (Herbert, C. in Chemical Engineering 146) to the gel. The stability of such a system was poor due to a lack of covalent or high affinity binding between the fibrin and the bioactive molecule.
Very little work has been done in incorporating peptide sequences and other bioactive factors into fibrin gels and even less has been done in covalently binding peptides directly to fibrin. However, a significant amount of energy has been spent on determining which proteins bind to fibrin via enzymatic activity and often determining the exact sequence which binds as well. The sequence for fibrin xcex3-echain crosslinking has been determined and the exact site has been located as well (Doolittle, et al., Biochem. and Biophys. Res. Comm., 44:94-100, 1971). Factor XIIIa has also been shown to crosslink fibronectin to fibronectin (Barry and Mosher, J. of Biol. Chem., 264:4179-4185, 1989), as well as fibronectin to fibrin itself (Okada, et al., J. of Biol. Chem., 260:1811-1820, 1985). This enzyme also crosslinks von Willebrand factor (Hada, et al., Blood, 68:95-101, 1986), as well as xcex1-2 plasmin inhibitor (Tamaki and Aoki, J. of Biol. Chem., 257:14767-14772, 1982), to fibrin. The specific sequence that binds from xcex1-2 plasmin inhibitor has been isolated (Ichinose, et al., FEBS Letters, 153:369-371, 1983) in addition to the number of possible binding sites on the fibrinogen molecule (Sobel and Gawinowicz, J. of Biol. Chem., 271:19288-19297, 1996) for xcex1-2 plasmin inhibitor. Thus, many substrates for factor XIIIa exist, and a number of these have been identified in detail.
The present invention in a general and overall sense, provides unique fusion proteins and other factors, either synthetically or recombinantly, that contain both a transglutaminase domain such as a Factor XIIIa substrate domain and a bioactive factor, these peptides being covalently attached to a fibrin substrate having a three-dimensional structure capable of supporting cell growth.
In some embodiments of the present invention, bioactive properties found in extracellular matrix proteins and surface proteins are confined to a structurally favorable matrix that can readily be remodeled by cell-associated proteolytic activity. In some embodiments, the fibrin is gel matrix. A bioactive means is also included to facilitate the incorporation of an exogenous signal into the substrate. In addition to retaining the bioactivity of the exogenous signal molecule, the overall structural characteristics of the fibrin gel is maintained.
The invention in another aspect provides for a fibrin matrix comprising short peptides covalently crosslinked thereto, as well as bioactive factors. The fibrin matrix may be further defined as a fibrin gel. The matrix chosen is fibrin, since it provides a suitable three dimensional structure for tissue growth and is the native matrix for tissue healing. It is anticipated that other, fibrin-like matrices may also be similarly prepared. The crosslinking was accomplished enzymatically by using the native Factor XIIIa to attach the exogenous factors to the gels. In order to do this, a sequence that mimics a crosslinking site was incorporated into the peptide so that the enzyme recognized and crosslinked it into the matrix. Novel activity will be conferred to these fibrin gels by adding a peptide sequence, or other bioactive factor, which is attached to the crosslinking sequence. These materials may be useful in the promotion of healing and tissue regeneration, in the creation of neurovascular beds for cell transplantation and in numerous other aspects of tissue engineering. Hence, the invention in yet other aspects provides compositions created and adapted for these specific uses.
The following sequences are referenced throughout the Specification:
In one aspect, the invention provides a composition that comprises a protein network and a peptide having an amino acid sequence that comprises a transglutaminase substrate domain and a bioactive factor (e.g., peptide, protein, or fragment thereof) is provided. The peptide is covalently or at least substantially covalently bound to the protein network. In particular embodiments, the protein network is fibrin or a fibrin-like molecule. In other particular embodiments, the transglutaminase substrate domain is a factor XIIIa substrate domain. This factor XIII, a substrate domain may be further defined as comprising an amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a fragment thereof, a combination thereof, or a bioactive fragment of said combination. Some embodiments may be defined as comprising a bioactive factor that comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, a fragment thereof, a combination thereof, or a bioactive fragment of said combination.
In another aspect, the invention provides an implantable device having at least one surface or portion of at least one surface that comprises the composition of any one of the above compositions described herein. By way of example, the implantable device may be fashioned as an artificial joint device, such as a knee replacement. The invention may also take the form of a porous vascular graft, wherein at least one region or a portion of at least one region of the porous vascular graft comprises a porous wall that includes the composition of the protein network and covalently attached peptide/protein described herein. The invention as a device may be further defined in other embodiments as a scaffold for skin, bone, nerve or other cell growth, comprising a surface that includes at least one region or area that comprises the composition of the protein matrix and covalently attached peptide described herein.
In yet another aspect, the invention provides for a surgical sealant or adhesive comprising a surface that includes the composition of the peptide matrix and covalently attached peptide on at least one region of the surface.
The invention further provides methods for promoting cell growth or tissue regeneration. This method comprises in some embodiments, covalently attaching or producing a covalently attached bioactive complex molecule comprising a bioactive factor and a transglutaminase substrate, covalently coupling the bioactive complex molecule to a peptide network capable of having covalently attached thereto the bioactive factor or a fragment thereof, to provide a treated peptide substrate; and exposing said treated peptide substrate to a composition comprising cells or tissue to promote cell growth or tissue regeneration. This method may be used in conjunction with a variety of different cell types and tissue types. By way of example, such cell types include nerve cells, skin cells, and bone cells. The peptide network may be further defined as a protein network, such as a fibrin network. The transglutaminase substrate may be further defined as a factor XIIIa substrate, while the transglutaminase may be further defined as factor XIIIa. The factor XIIIa substrate may be further defined as having an amino acid sequence of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, a fragment thereof, a combination thereof, or a bioactive peptide fragment of said composition. The peptide may, in some embodiments, be further defined as comprising an amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, a fragment thereof, a combination thereof or a bioactive peptide fragment thereof.
The invention in yet another aspect may be defined as a biosupportive matrix material. This material in some aspects, comprises a peptide network and a bioactive factor, wherein said bioactive factor is covalently attached to the peptide substrate. This peptide substrate may be further defined as a protein network. The bioactive factor is covalently attached to the substrate through a transglutaminase or a similar enzyme. The peptide that may be used in conjunction with the invention may comprise any variety of peptides capable of being covalently attached to the fibrin substrate or biosupportive matrix as described herein. In some embodiments, the peptide may be further defined as comprising an amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 10, a fragment thereof, a combination thereof, or a bioactive fragment thereof.
In particular embodiments of the matrix compositions, the calculated moles of peptide that is to be included may be defined or described for those devices/surfaces that include them, as virtually any amount of peptide that falls within a physiologically relevant concentration of the particular peptide/protein selected. For a standard gel, 1 mg of fibrinogen would typically be included. Hence the concentration of fibrinogen in this standard gel may be described as about 3xc3x9710xe2x88x926 mM. Using this figure as a benchmark in one example, the ratio of the amount of peptide to fibrinogen could be expressed as about 3xc3x9710xe2x88x926 mM to about 24xc3x9710xe2x88x926 mM.